Specific communication between enhancers and alternative promoters mediates finer regulation of transcription initiation
- Author(s): Bardales Mendieta, Jorge Adalberto;
- Advisor(s): Darzacq, Xavier;
- et al.
Enhancers are DNA regulatory elements that play an important role in the precise
regulation of Pol II transcription initiation and, consequently, in the generation and
maintenance of patterns of gene expression. A defining characteristic of enhancers is their
capacity to regulate transcription from their target promoter, independent of the genomic
distance that separates them. Importantly, in order for enhancers to exert their regulatory
action, they must first relocalize into close spatial proximity to their target promoter and
form long-range interactions. Although these interactions have been demonstrated to
occur, the mechanisms that allow their specific and efficient formation remains largely
obscure. In this thesis, I focus on the development of tools and the expansion of our
understanding with regard to this important topic.
In the first chapter, I begin by providing a perspective of transcription initiation as
a hub of gene regulation. Then, I perform a concise overview of promoters and enhancers
as DNA regulatory elements and share the most important functional and structural
characteristics. I finalize the chapter by discussing the known regulatory mechanisms that
allow the formation of specific interactions between enhancers and promoters to take place.
In the second chapter, I present my findings on the development of imaging systems
to permit the visualization of enhancer-promoter interactions in live cells. I begin by
describing the limitations of current systems. Then, I discuss the importance of the
development of novel, innovative systems that allow specific labelling of genomic loci.
From there, I present the possibilities and limitations of a developed dCas9/Aptamer
system for the visualization of repetitive and non-repetitive loci. I also present my findings
on the development of a two-step integration system of operator arrays and share the
principal technical limitations encountered in its development. I finish by discussing and
providing a critical perspective of relevant published work that has been produced since
these efforts were undertaken.
In the third chapter, I begin by identifying and characterizing potential enhancers
in the PCSK9 locus in hepatic derived cells. I identify three candidate enhancers with
strong transcriptional activity by reporter assays. Then, I demonstrate how the regulatory
activity of these candidate enhancers is specific to hepatic cells and their regulatory activity
can be modulated by two small molecules in a similar fashion to the endogenous PCSK9
gene. From there, I dissect these DNA regulatory sequences to identify transcription factor
binding sites responsible for transcriptional activity by bioinformatic analysis and fine
mutagenic studies. These results point towards the presence of three DNA regulatory
elements required for PKCS9 expression.
In the fourth and final chapter, I investigate two alternative promoters (P1 and P2)
that drive MYC expression and are regulated by enhancers. The two MYC promoters can
be differentially regulated across cell-types, and their selective usage is largely mediated by
distal regulatory sequences. Moreover, in colon carcinoma cells, Wnt-responsive enhancers
prefer to upregulate transcription from the P1 promoter, using reporter assays in the
context of the endogenous Wnt induction. In addition, multiple enhancer deletions using
CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, I explore how
preferential activation between Wnt-responsive enhancers and the P1 promoter is
influenced by the distinct core promoter elements that are present in the MYC promoters.
Taken together, these results provide new insight into how enhancers can specifically target
alternative promoters and suggest that the presence of alternative promoters could create a
more efficient formation of long-range interactions and a more precise combinatorial
regulation of transcription initiation.